Intercellular communication mediated by gap junctions is essential for the synchronized spread of excitation between myocardial cells. I propose to study the biochemistry and biophysics of the cardiac gap junction channel. I will use cDNA for rat liver gap junction protein, which I have already cloned and characterized, as a probe to isolate cDNA for rat cardiac gap junction protein. The primary sequence of the heart gap junction will be determined and compared to liver. Antibodies specific to defined protein domains will be produced and used to determine, by immunocytochemical methods, the transmembrane orientation of the channel protein. The channel forming ability of the protein will be studied by using the cloned cDNA to produce molecularly pure heart gap junction protein in vitro. The protein will be incorporated into membrane vesicles by translation of synthetic mRNA in the presence of dog pancreatic microsomes and then fused into planar lipid bilayers where the ion selectivity and gating properties of the channel can be precisely determined. I will attempt to develop a system where the requirements for the establishment of gap junction-mediated intercellular communication can be examined. Xenopus oocytes will be injected with synthetic RNA. The synthesis, processing, membrane insertion and assembly of gap junction protein will be examined. The ability of pairs of similarly injected oocytes to form functional gap junctions will be assessed. These studies will provide a molecular basis for the understanding of synchronization between heart cells, and the pathogenesis in synchronization patterns which accompany cardiac ischemic and reperfusion episodes.